Re: Permeabilization with ethanol (Whew! What a mouthful)

A 16:45 08/11/1999 EST, Nperson211@aol.com a écrit :
>Histonetters,
>I am looking for a reference for using buffered, graded ethanols for
membrane
>permeabilization that is a little less destructive than saponin or other
>methods. A detailed method would suffice or the exact reference you use.
Any
>and all replies will be appreciated.
>Thanks in advance,
>Nancy Lemke
Hi,
I have used buffered ethanol membrane permeabilization for ICC with very
good results, at least in terms of permeabilization. I don't know whether
it is less destructive than saponin. Ethanol being a precipitating
fixative, ethanol permeabilization may indeed precipitate some membrane
proteins (or even cytoplasmic proteins) that would be extracted by the use
of detergents as saponin.
We use a procedure derived from the following paper:
Eldred WD et al. (1983) J Histochem Cytochem 31 (2): 285-292
I have never looked at the ultrastructure of the tissue, which according to
these authors should be quite OK. What I know is that this procedure is
much more efficient than detergent permeabilization in ICC. I checked 100
micron thick tissue slabs (wouldn't call those "sections") by embedding
them in resin and cutting them transversely and the immunolabel was clearly
present throughout the whole thickness of the sections.
We use the following procedure:
Make up 10, 20 and 40% ethanol in 100 mM phosphate buffer. The 20 and
especially the 40% ethanol have to be pH adjusted. Also, the phosphate
tends to precipitate in the 40% ethanol, especially if you cool it down.
To permeabilize, treat the sections in sequence 10, 20, 40, 20, 10 and
finally buffer for 5 to 10 minutes each.
Paul
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Paul Klosen, PhD
CNRS UMR 7518 Neurobiologie des Fonctions Rythmiques et Saisonnieres
Universite Louis Pasteur 12, rue de l'Universite
F-67000 Strasbourg, FRANCE
Tel. 03.88.35.85.04 Fax. 03.88.24.04.61
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